Our work on Adeno-associated virus (AAV)vectors has focused on the basic biology of AAV DNA replication and capsid assembly. This has lead to important information that has allowed the development of new vector production strategies and the promise of targeted vectors. In this application, we propose to continue this work by focusing on three problems related to viral DNA replication and packaging: 1) Identification of essential components of AAV DNA replication. This aim will determine which of the proteins associated with the MCM complex (MCM2-7) are required for in vitro AAV DNA replication. It has previously been established that MCM 4, 6 and 7 form a minimal complex that contains DNA helicase activity. We wish to establish if this minimal complex alone is capable of AAV leading strand DNA synthesis, or if other components, e.g. MCM2,3,5 and 10, CDC45, cdtl, GINS and Cdc6 are also required for AAV DNA replication. Additionally no studies have shown directly that MCM helicase activity is involved in leading strand AAV DNA synthesis. In this aim we plan to determine by mutagenesis if the helicase activity of MCM is essential for AAV DNA replication. 2) The identification of protein interacting partners in DNA replication and packaging and the mechanism of strand elongation. We will use defined substrates and the essential purified components required for AAV DNA replication to ask basic questions about the mechanism of AAV DNA replication. These include whether MCM loading requires Rep protein, whether the pol 6 complex maintains contact with Rep and MCM during strand elongation, and what the role of the Rep-capsid complex in DNA replication and packaging. Finally, we intend to identify the specific interacting partners in DNA replication using electron microscopy and cryo-EM. 3) Mapping the position of the AAV Rep proteins on the surface of the AAV capsid. It is now well established that Rep is the packaging signal and provides the helicase motor for driving AAV DNA into the capsid. We will use cryo-EM, cryo-tomography and X-ray crystallography to determine the interaction site between Rep and the capsid that is believed to initiate packaging. RELEVANCE (Seeinstructions): Recombinant Adeno-associated virus vectors (rAAV) are efficient and safer gene transfer vehicles that show enormous promise for gene therapy. This proposal focuses on the basic biology of AAV DNA replication and viral packaging. Our hope is that the information obtained will enable us to improve production methods, find ways of creating improved vectors that will become the next generation of gene therapy transfer vehicles.